Microarray fabricating device

ABSTRACT

A device for fabricating a high-density microarray for cDNA or protein having an arbitrary pattern comprises an electrospray part for electrostatically spraying solutions containing a plurality of kinds of biologically active samples one by one, a support part supporting sample chips on which samples in the solutions sprayed from the electrospray part are deposited, a mask part disposed between the electrospray part and the support part and having holes the number of which is the same as that of the sample chips so as to selectively deposit the samples simultaneously in the adequate positions corresponding to the sample chips, a moving part for fabricating microarrays at a time by relatively moving the sample chip support part and the mask part and depositing the samples on the sample chips. Therefore the device can fabricate a large number of inexpensive high-density microarrays.

FIELD OF THE INVENTION

The present invention relates to a device (Microarrayer) formanufacturing microarrays (such as DNA chips, protein chips and organiccompound chips, etc.).

BACKGROUND ART

A genome of kinds of bacteria and yeast (i.e. base sequence of theentire genes) has been determined in late years, and a human genomewould be determined completely in the near future. Such a rapid progressof the genome sequencing technology makes it possible to clarifyfunctions of the determined genes and functions of proteins derived fromthe determined genes. It is said that the number of genes of yeast isapproximately 6,200 and the number of genes of human being isapproximately 100,000. Thus, a technology for manipulating at the sametime a vast number of genes such as proteins and others is needed formaking clear these functions. The microarray technology has been rapidlydeveloped in these years for accomplishing the above object and hasattracted great attentions. The object of this technology is to achieveexperimental systems for synthesizing a number of oligonucleotides onsubstrates such as slide glasses and for immobilizing cDNAs or proteins.For example, there has been developed an experimental system, in which alarge number spot of cDNAs of all genes (genome) are disposed on onepiece of slide glass, these spots are hybridized, and an intensity ofthe hybridization of respective spots is measured to determineexpressions of respective genes.

For instance, U.S. Pat. No. 5,445,934 discloses a DNA chip includingsynthesized oligonucleotide on a substrate at a rate of not less than1,000 spots/cm². Furthermore, “Nature Genetic Supplements”, Vol. 21(Patrick O. Brown et al. p33–37; David D. L. Bowtell p25–32, 1999,January) discloses a method for spotting cDNA solutions on a slide glassusing a pin. Also, U.S. Pat. No. 5,807,522 discloses a method forspotting cDNA solutions on a slide glass using a solenoid vibrating thesolutions.

There have been proposed the following methods of manufacturingmicroarrays:

-   (1) Photo-lithography method-   (2) Micro spotting method-   (3) Ink-jet method.

In the method (1), oligonucleotides are synthesized on a substrate usingthe same photo-lithography technology as that employed for manufacturinga semiconductor device. In the method (2), solutions of cDNAs and thelike are spotted on a substrate using a pin-like tool. In the method(3), solutions of cDNAs, etc. are dropped from a narrow nozzle using apiezoelectric transducer and so on.

According to the method (1), successive spots can be placed at aninterval of about 50–25 μm to manufacture a microarray having a highspot density. However, in this method, since an oligonucleotide issynthesized on the substrate, this method could not be applied to cDNAswhich have been prepared separately. In addition, it takes a long timeto design and manufacture a photomask, and thus this method is veryexpensive. In the methods (2) and (3), while these methods can beapplied to cDNAs which have been prepared separately, a diameter ofresulting spots is relatively large such as approximately 300–150 μm,and therefore, it is hard to manufacture a microarray with a high spotdensity. Owing to the reason that these methods require mechanicallyoperation, they are suitable for manufacturing chips in small quantity,but are not suited for manufacturing chips in mass production. Accordingto a document (Vivian G. Cheung et al. “Nature Genetic Supplements”1999, January), if a size of a spot is reduced from 200 μm to 50 μm, aquantity of sample needed for making a chip goes down to about 1/100.Thus, upon realizing a practical microarray, one of critical problems tobe solved is that how to reduce a spot size in order to obtain a chipwith a high spot density.

In order to make clear functions of various genes or proteins and to usethese findings in researching new drugs, diagnosing diseases andselecting optimal drugs for individual persons and so on, a microarraycontaining cDNAs or proteins must be manufactured with a small spot sizeand a high spot density at low cost. Therefore, the present invention isto provide a device for manufacturing a high spot density microarrayhaving a spot size (i.e. diameter) of several μm to several tens μm byusing one or more samples which have been prepared separately.

In PCT international publication WO98/58,745 and a document “AnalyticalChemistry”, Vol. 71′ (Morozov et al. pp. 1415–1420, pp. 3110–3117,1999)′, there have been proposed a device and a method for making solidspots or a film on a substrate by using the electrostatic spray, whilethe biologically activities of biomolecules such as a nucleic acid or aprotein has are retained. A method and a device for manufacturing amicroarray with very small spots simultaneously by adjusting variousconditions have been also disclosed. However, since these method anddevice use a filter in the form of mesh, they could not provide amicroarray, in which various samples are located at desired positions.

SUMMARY OF THE INVENTION

The present invention has for its object to provide a device formanufacturing a high spot density microarray, in which one or more cDNAsor proteins are arranged in accordance with an arbitrarily pattern bydeveloping the above mentioned knowledge. According to the invention, adevice for manufacturing microarrays comprises:

-   -   electro-spraying means for electrostatically spraying, in        sequence, a plurality of solutions each containing respective        one of a plurality of kinds of biologically active samples;    -   supporting means for supporting a plurality of sample chips on        which samples contained in the solutions and electrostatically        sprayed from said electro-spraying means are deposited;    -   masking means disposed between said electro-spraying means and        said supporting means and having holes whose number is equal to        the number of the sample chips, a sample being selectively and        simultaneously deposited on said sample chips at predetermined        corresponding locations; and    -   shifting means for shifting relatively said supporting means and        said masking means such that the samples are deposited on said        plurality of sample chips to manufacture simultaneously a        plurality of microarrays. Upon using the device for        manufacturing microarrays according to present invention, a        capillary provided in the electro-spraying means is moved to a        center of an electro-spraying region and is connected to a high        voltage power source, and then the method disclosed in said        patent publication and document (WO98/58,745 and “Analytical        Chemistry Vol. 71”) is carried out to perform the electrostatic        spray.

In a first embodiment of the device for manufacturing microarraysaccording to the invention, said electro-spraying means comprises asingle capillary including one or more electrodes and liquid supplyingmeans for feeding said plurality of solutions to said single capillaryin sequence, each of said solutions containing respective one of saidplurality of samples. According to need, the device may further comprisecleaning means for washing the capillary after a solution iselectrostatically sprayed and before a next solutions is to be sprayed.

In a second embodiment of the device for manufacturing microarraysaccording to the invention, said electro-spraying means furthercomprises holding means for holding a plurality of multi-capillarycassettes each of which includes a plurality of capillaries each havingone or more electrodes which are selectively connected with a powersource for electro-spraying; and conveying means for conveyingsuccessively said plurality of multi-capillary cassettes to anelectro-spraying location. In both of the first and the secondembodiments of the microarray manufacturing device, saidelectro-spraying means may further comprise pressurized air supplyingmeans for supplying a pressurized air to a single capillary or all ofthe capillaries in said multi-capillary cassettes simultaneously toconvey the solution to a tip of the capillary or tips of all capillariesupon the electro-spraying. Furthermore, in both of the first and secondembodiments of the microarray manufacturing device, the device maycomprise driving means for moving the single capillary or themulti-capillary cassette upon the electro-spraying.

In order to assist the electro-spraying, the device may comprisepressurized air supplying means for supplying simultaneously apressurized air to all the capillaries in said multi-capillary cassettesto convey the solution to tip of the all capillaries when the solutionis electrostatically sprayed by the electro-spraying means. Moreover,said holding means for holding a plurality of multi-capillary cassettesmay includes means for controlling temperature of a plurality ofsolutions contained in the capillaries in the multi-capillary cassettes(for example cooling them). According to this arrangement, thebiological activities or biological functions of samples can be keptmuch more effectively.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view illustrating the arrangement of the singlecapillary system according to the invention;

FIG. 2 is a perspective view depicting the arrangement of themulti-capillary system according to the invention;

FIG. 3 is a cross section diagram and an exploded perspective view of amask;

FIG. 4 is a perspective diagram illustrating the structure of themulti-capillary cassette;

FIG. 5 is a perspective diagram showing the structure of the singlecapillary system;

FIG. 6 is an electrical circuit diagram depicting the multi-capillarysystem;

FIG. 7 is a schematic diagram showing the electrical wiring and pipelayout;

FIG. 8 is a schematic diagram illustrating the driving mechanism duringthe manufacture of the microarrays and X-Y system and X-Y-Z system; and

FIG. 9 is a schematic diagram depicting a manner of moving a mask on X-Yplane as well as a sequence of forming a number of spots.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS First Embodiment

A first embodiment of the device for manufacturing microarrays accordingto the present invention constructed as a single capillary system willbe described. As shown FIG. 1, the single capillary system comprises asingle capillary 11. The system is mainly consisting of anelectro-spraying part 10, a masking part 20 and a supporting part 30 forsupporting a substrate. In the device for manufacturing microarrays ofthe present embodiment, a plurality of the solutions containingrespective biological active samples are electrostatically sprayed andare deposited on desired positions in depositing areas of substrates bymeans of a mask. This system comprises the capillary 11 including anelectrode, the electro-spraying part 10 including a guard ring 12 and ashield 13, the masking part 20 including a mask 21 and a mask holder 22,and the movable substrate supporting part 30 including a plurality ofsample chips 31 and a sample chip holder 32. That is, the device formanufacturing microarrays according to the invention compriseselectro-spraying means for electrostatically spraying, in sequence, aplurality of solutions each containing respective one of a plurality ofkinds of biologically active samples; supporting means for supporting aplurality of sample chips 31 on which samples contained in the solutionsand electrostatically sprayed from said electro-spraying means aredeposited; masking means disposed between said electro-spraying meansand said supporting means and having holes whose number is equal to thenumber of the sample chips 31, a sample being selectively andsimultaneously deposited on said sample chips at predeterminedcorresponding locations; and shifting means for shifting relatively saidsupporting means and said masking means such that the samples aredeposited on said plurality of sample chips to manufacturesimultaneously a plurality of microarrays.

The capillary 11 (made of glass, plastics, etc.) may contain solutionsincluding cDNA, proteins, etc., and has the electrode provided therein.The capillary 11 is configured to apply a voltage to the solution viathe electrode and a pressurized air is supplied to the capillary fromits top when required. In case of spraying many kinds of cDNAs, proteinsand the like, capillaries can be selectively mounted by using acapillary changer (not shown in FIG. 1) or manually. Alternatively, inorder to prevent contamination, the capillary 11 may be washed by purewater and the like each time solutions are changed during theelectro-spraying. The device may comprise means for moving one or morecapillaries during the electro-spraying (not shown in FIG. 1). By movingthe capillary during the electro-spraying, a sample can be sprayed overa wider area in one-spray, that is a samples can be spread over a largernumber of sample chips in one-shot. Said moving means may be constructedsuch that the capillary is shifted or the sample holder and/or mask maybe shifted. That is to say, the moving means may be constructed to movethe capillary and the sample chips relatively. The guard ring 12 servesas an electrode for preventing diffusion of sprayed particles (i.e.solution) and is made of an electrically conductive material. An ES(Electro-Sprayer) is fully covered with a casing 14 such that the entireelectro-spraying part can be protected from an influence of moisture andair turbulence. The shield 13 made of an electrically insulatingmaterial or a dielectric material (such as glass and plastics) andfunctions to spread sprayed particles uniformly.

The casing 14 has a dry air inlet 15 for supplying a clean and dry gassuch as a dry air to accelerate a drying speed of sprayed particles andto suppress contamination by ambient atmosphere. The sample chip holder32 supports a plurality of sample chips 31 (i.e. microarrays) such thatthe sample chips 31 are fixed by evacuation or electrostatic attractionto keep a right position relative to the mask 21. The sample chip holder32 is precisely disposed in parallel with the mask 21 to keep a distance(a gap) between the holder 32 and the mask 21 constant. An X-Y stage 33is controlled such that the holder 32 is driven over an X-Y plane tovary a relative position between the chips 31 and the mask 21 to formspots of samples at desired positions.

A Second Embodiment

A second embodiment of the device for manufacturing microarraysaccording to present invention constituted as the multi-capillary systemwill be described. As shown FIG. 2, the multi-capillary system usesmulti-capillary cassettes each having a plurality of capillaries 51 andis suitable to spray more kinds of cDNAs or proteins effectively thanthe above explained single capillary system, while different kinds ofsolutions can be electrostatically sprayed without cross contaminationby automatically changing the capillaries 51. A plurality of capillaries51 are held in a cassette 52, and each cassette comprises a connector 53connected to electrodes provided in respective capillaries 51 viaelectrical wiring and a gas supply channel for applying pressure. Byusing these components, an ESD (Electrostatic Spray Device) electricunit 54 (high voltage generating and switching) connected to theconnector 53 through a cable 55 applies a high voltage to thecapillaries 51 selectively to select samples to be electrostaticallysprayed rapidly and many kinds of spots can be deposited rapidly.

When a multi-capillary cassette 52 is mounted, electrodes provided incapillaries within the cassette can be connected to the high voltagepower supply source 54 via the connector 53 and cable 55. The ESD unit54 generates a high voltage required for electro-spraying and changesthe application of the high voltage to capillaries to alter substancesto be electrostatically sprayed. In addition to the high voltage (about2000–4000 V) to be provided to solutions to be electrostaticallysprayed, the ESD unit 54 also provides high voltages (about 500–3000 V)applied to a guard ring and a collimating ring (not shown FIG. 2).

The case 56 protects an electro-spraying process from disturbance likeas the single capillary system. A large-scaled shield 57 is formed by amesh of an insulating material or a dielectric material and ensuresuniform distribution of the electrostatically prayed particles. An autocapillary changer 58 is constructed by a robot arm or an X-Y-Z stage andis movable freely between an electro-spraying part and a multi-capillarycassette hanger 59. Thus the changer 58 can exchange the multi-capillarycassettes. Sample solutions may be supplied into capillaries by means ofa filler equipped in at hanger 59 or may be sucked into capillaries froma sample pallet 60 provided separately by means of an automatic sampler.

In the multi-capillary system, although relative positions of respectivecapillaries and corresponding masks and substrates are different, if adistance between the capillary and the mask or substrate is long enough,particles would be diffused uniformly and are deposited uniformly atdeposition areas on respective substrates. In a modified embodiment, thecapillaries are successively indexed at a center of the electro-sprayingpart each time respective solutions are sprayed. Like as the singlecapillary system, also in the multi-capillary system there may bearranged the driving means for moving the capillary upon theelectro-spraying (not shown in FIG. 2).

A mask structure 40 shown in FIG. 3 has function for convergingparticles sprayed from the capillary and directing said particles toeach of spots to form desired size spots on desired respectivelocations. The mask 40 is applicable commonly to both the singlecapillary system and the multi-capillary system. The mask 40 is formedby laminating an insulating layer 41, an electrically conductive layer42, an insulating layer 43 and a mask layer 44 (insulating materiallayer) in this sequence viewed from the capillary. At an early stage ofthe electro-spraying, the insulating layer 41 is charged by depositionof charged particles. However, after that, charged particles areprevented from being deposited on mask layer and the insulating layer 41serves to converge the sprayed substances toward minute holes formed inthe mask 40. To this end, the mask structure 40 is designed such that adiameter of an opening of a hole facing the electro-spraying means has alonger diameter than that of an opening of the hole facing the samplechips (on a side of the substrate supporting part). The electricallyconductive layer 42 constituting the collimating ring is made of anelectrically conductive material such as metal. The conductive layer 42generates a electric field which repels the charged particles andcollects the particles toward centers of the minute holes by applying amedium voltage so that capturing efficiency is improved. The insulatinglayer 43 has a function for insulating the collimating ring from a masklayer 44 mentioned below.

The mask layer 44 is formed by a thin layer (for example range from tenμm to several hundred μm) of an insulating or dielectric material suchas mica and silica glass. The mask layer 44 has a number of perforatedholes 44 a (pore size in a range of a few μm to one hundred μm) whosesize (i.e. diameter) is almost same as that of a desired spot. It isconsidered that the mask layer has a similar function to that of theinsulating layers 41 and 43. That is to say, after the mask layer havebeen charged by the deposition of charged particles, charged particlesare directed to center of the hole by electrostatic repelling force. Aspacer 45 made of an insulating material such as plastics and glass or ametal and has a thickness of approximately several μm to several tensμm. The spacer 45 is provided for making a space between the maskstructure and the sample chips (i.e. the sample holder) to avoidmechanically contacting. The mask structure 40 has several tens toseveral ten thousands mask holes. By using such mask structure, a greatlarge number of sample chips can be manufactured simultaneously.

In general, sample chips whose number is equal to that of the holes ofthe mask structure are set and a plurality of chips are manufacturedsimultaneously in one task. The sample chip is made of optical glasscoated with an electrically conductive material (such as ITO (Indium TinOxide or a metal film etc.)), a metal plate, soda glass or electricallyconductive plastics. It should be noted that although plastic materialsare considered to be electrically insulating, they have a slightconductivity, in general, and therefore plastic materials may be used asa substrate without applying an electrically conductive coating. Thus ina practical sense materials which are not suited for the sample chip islimited to fluorocarbon resin, silica glass and so on. An electricallyconductive part of the chip is grounded through, for instance, thesample holder in order to remove an electrostatic charge generated bycharged particles deposited on the chips. In the present embodiment, thechips are shifted to adjust deposition sites of particles. Instead ofmoving the chips, the mask may be moved to adjust the depositionlocations. Also, instead of moving the mask, deposition sites on chipsmay be shifted by forming a substrate by a transparent glass coated witha photo-conductive layer and by irradiating light from beneath of theglass chips. It should be noted that a chip size, the number of thechips and so on may can be varied in various ways. For example, thefollowing arrangement may be used in this embodiment:

-   -   chip size; 10 mm by 10 mm    -   the number of chips manufactured in one time; from one hundred        (10×10) to several thousands (33×33),    -   the number of spots: from 1,000 to 100,000,    -   spot size; circular shape having a diameter of about 10–50 μm    -   spot pitch; from 20 μm to 100 μm.        It is easy to enlarge a spot size, but in this case a chip size        has to be increased or the number of spots has to be reduced.        According to the invention, sample substances are, in general,        proteins such as enzyme, refined receptor, monoclonal antibody,        fragment of antibody. Also, DNA and its fragment, cDNA and its        fragment, various kinds of organic macromolecule compounds, and        minute particles such as membrane integral receptor and virus        may be also used as sample substances.

In the present embodiment, since the mask with 100 holes is utilized,100 (10 by 10) chips (microarrays) are set on the sample holder. Amulti-capillary cassette with 96 wells is used as a capillary storage,and each capillaries have different sample solutions. Now it is assumedthat 10,000 spots are to be formed in total, 105 units ofmulti-capillary cassettes each having 96 wells and 1,000 kinds of samplesolutions have to be prepared. Each of these solutions is infused intoeach of the capillaries. A square plate of 10 mm by 10 mm is used as asample chip and spots having a diameter of 20 μm are to be depositedwith a pitch of 80 μm. In this manner, 10,000 spots can be formed on asingle chip. A deposition time of about 10 seconds is required to formone spot, and therefore it takes 28 hours to form 10,000 spots. In thisembodiment, since 100 units of chips can be manufactured simultaneously,100 units of chips each having 10,000 spots can be manufacturedsimultaneously in 28 hours.

The mask structure is designed to be manufactured easily in thefollowing manner:

-   (1) A metal layer (aluminum, copper, etc.) and an insulating layer    are successively provided on an insulating plate (PMMA, fluororesin,    etc.).-   (2) The thus laminated plate is drilled from above using a tool such    as end mill and a number of conical holes are formed.-   (3) A number of minute holes are formed in a plate made of mica or    silica glass, etc. using abrasive jets, etching or microscopic    machining or the like, then this plate is built-up on the laminated    plate formed in the process (2).-   (4) Finally the spacers are adhered on a bottom surface of the    laminated plate. In this manner the mask structure is attained.

As shown in FIG. 4, a multi-capillary cassette 70 includes a capillarysupport base 71 (made of plastics such as PMMA, etc.), and a pluralityof capillaries 72 are mounted on the base 71. Each of the capillaries 72has an electrode (not shown) and each of the electrode is connected withan electric connector 73 via a wiring pattern 74. These capillaries 72contain different kinds of sample solutions. The multi-capillarycassette further comprises a pressurized air inlet 75 and conduitchannels 76. Upon electro-spraying, it is possible to pressurize thesample solutions contained within the capillaries, and when the inlet iscoupled with a suction device, it is possible to suck in the solutionswithin the capillaries. In the present embodiment, the application ofpressure and reduced pressure is carried out for all the capillariessimultaneously. By applying the pressure, a sample solution is fed to atip of capillary to make a condition that the electro-spraying could beperformed easily. This application of pressure is not essential and issecondarily used for making a condition that the spraying is performedeasily. In this manner, under the situation that the sample solutionsare fed to tips of all capillaries, a high voltage is applied to one ofthe capillaries, and then a sample solution contained in this capillaryis sprayed by the electrostatic force to forms a number of minutedroplets. Of course, it is also possible to apply the pressure only to aselected one capillary and a high voltage is applied to this selectedcapillary. Usually the multi-capillary cassette 70 is disposable andthere is no need for washing. The multi-capillary cassette may be alsoreused by washing. A number of sample solutions are simultaneouslysucked into capillaries from the sample palette, as shown in FIG. 2.Alternatively, capillaries having sample solutions previously containedtherein may be mounted on the base.

FIG. 5 shows a single capillary 80, which comprises a capillary 81(about one to several mm in diameter) with a narrow tip (about a few μmto several tens μm in diameter) made of glass or plastics, etc., anelectrically conductive wire 82 (made of platinum, etc.) as a electrodeand a capillary holder 83. The capillary 81 contains a sample solution.The capillary holder 83 is connected with the high voltage power supplythrough the conductive wire 82. At a top of the capillary holder 83there is provided an inlet 84 for introducing the pressurized air whichassists the elactro-spraying or the reduced pressure to suck a samplesolution from a tip of the capillary. Since different kinds of samplesolutions are used, capillaries are exchanged for respective kinds ofsolutions, alternatively a single capillary can be reused by sucking anddischarging a pure water. However, since the screening experiment allowsslight contamination, the capillary exchange and the capillary cleaningare not necessary.

FIG. 6 illustrates an electrical connection of the multi-capillarysystem. In the multi-capillary system, there are provided a plurality ofcapillaries 90, a high voltage switch 91 (provided within the electricequipment), high voltage power supplies V1, V2 and V3, a guard ring 92,a mask structure 93 and a substrate supporting part in which a number ofsample chips 94 are arranged. The sample chip 94 is coated with anelectrically conductive material or is made of an electricallyconductive material, and its conductive part is connected to 0 V (groundpotential). The mask structure 93 is located just above the sample chips94. A guard ring electrode 95 is connected with the collimating voltagepower source V3. The guard ring 92 is connected with the voltage powersource V2. The electro-spraying voltage power source V1 is connected toa capillary 90 via the high voltage switch 91. These voltages aregenerally set to V1=2,000–5,000 volt., V2=2,000–5,000 volt. andV3=500–3,000 volt., and have relationship of V1·V2>V3. In accordancewith the switching of the high voltage, the mask structure 93 is drivenby an X-Y stage (or X-Y-Z stage) to form sample spots with desired sizeat desired locations. By repeating this operation, a plurality of thechips having a desired large number of spots with desired size can bemanufactured simultaneously.

FIG. 7 is a schematic diagram showing the electrical wiring and conduitlayout of the single capillary system. While said multi-capillary systemhas a plurality of the capillaries, the single capillary system has onlyone capillary 100. Various kinds of sample solutions are successivelyfed into the single capillary 100 to form microarrays. The singlecapillary 100 is coupled with a pump 101 and a sampler 102 for suckingthe sample solutions 103. In this manner, the sample solutions such ascDNA and protein can be successively fed into the capillary 100. Itshould be noted that since a diameter of a conduit tube 107 issufficiently small, sucked sample solutions are banked as respectivelayers within the tube and can be prevented from being mixed with eachother. In order to manufacture microarrays, the sample chips 104 or themask 105 is shifted using the X-Y (or X-Y-Z) stage every time samplesolutions are electrostatically sprayed such that sprayed particles aredeposited at desired positions to form sample spots. In case of thescreening experiment, slight contamination is no problem, and therefore,sample solutions can be sucked into capillaries without washing orexchanging capillary. Alternatively, a pure water may be sucked into thecapillary between successively sucked sample solutions, and afterspraying a sample solution, a pure water is sprayed to clean inner wallsof the capillary and conduit pipe. In this case, the pure water used forcleaning may be discharged by a pressurized air, and then the dischargedwater is easily vaporized. Thus, in general, it is unnecessary toprovide any means for collecting the water.

FIG. 8 is a schematic diagram illustrating the driving method of the X-Ystage or X-Y-Z stage during the formation of microarrays. That is, FIG.8 shows relative positions between the mask structure and the samplechips during the formation of sample spots. As described above, thespacers are mounted on the bottom surface of the mask structure forkeeping an appropriate distance between the mask structure and thesample chips to avoid undesired mechanical contact of the mask withdeposited spots of cDNA or protein and prevent contamination or damage.Therefore, the spacers contact with surfaces of the sample chips duringthe electro-spraying. A thickness of the spacer is determined by heightof a spot to be formed. The spacer is designed to have suitableconfiguration and dimension such that the spacer does not interferepreviously deposited sample spots. There are two driving methods; in thefirst method (FIG. 8A), the sample chips are moved only on the X-Yplane, and in the second method (FIG. 8B), the sample chips are moved onthe X-Y plane as well as in the Z-direction (a direction perpendicularto the mask). The first method is applicable if surfaces of the samplechips and spacers are made of materials having a relatively goodabrasion proof. Since it does not require the control in the Zdirection, the stage has a simpler structure. The second method ispreferable for a case, in which surfaces of the sample chip and/or thespacers might be damaged by the movement of the spacers.

The driving method on the X-Y plane (FIG. 8A) is as follows:

-   (1) In the beginning, the mask structure is located at a    predetermined position on the sample chips.-   (2) Spots are formed on the sample chips by the electro-spraying.-   (3) The sample chips are moved on the X-Y plane by driving the X-Y    stage which holds the sample chips, such that next spotting    positions of the chips are indexed.-   (4) Spots are formed on the thus indexed spotting positions by the    electro-spraying.-   (5) A necessary quantity of sample spots are formed by repeating the    steps (3) and (4).

The second driving method in the X-Y-Z directions (FIG. 8B) is asfollows:

-   (1) In the beginning, the mask structure is located at a    predetermined position on the sample chips.-   (2) Sample spots are formed on the sample chips by the    electro-spraying.-   (3) The sample chips are removed from the mask structure by driving    the Z stage.-   (4) The sample chips are moved on the X-Y plane by driving the X-Y    stage such that next spotting positions are indexed.-   (5) The sample chips are contacted again with the mask structure by    driving the Z stage.-   (6) Sample spots are formed on the sample chips by the    electro-spraying.-   (7) A necessary quantity of sample spots are formed by repeating the    steps (3) and (4).

In the present embodiment, the sample chips are mounted on the X-Y stageor the X-Y-Z stage, while the mask is fixed. According to the presentinvention, it is sufficient to change a relative position between thesample chips and the mask, and therefore either one of the sample chipsand the mask structure may be driven in the X axis and Y axis and Zaxis.

FIG. 9 is a schematic diagram depicting a manner of moving the mask onthe X-Y plane and successive steps of forming a number of sample spots.As shown in an upper left part in FIG. 9, the mask has a number ofminute holes viewed from above, and sample chips 110 are disposed belowthe mask. One of the chips 110 is shown in an enlarged scale, a spacer112 is mounted on a bottom surface of a portion of the mask structurehaving a minute hole 111. The movement in the vertical direction (i.e. Zdirection) has been explained with reference to FIG. 8. In order toavoid contamination and damage of the already deposited sample spots,the relative movement of the mask structure and sample chips has to becontrolled precisely. In the present embodiment, the mask structurehaving specially configured spacers 112 shown in FIG. 9 is utilized, andsample spots are formed in the following manner:

-   (1) The minute hole 111 in the mask is indexed at an upper left    corner of a sample chip 110.-   (2) A first spot is formed on the sample chip by the    electro-spraying.-   (3) The mask is sifted to right.-   (4) A second spot is formed on the sample chip by the    electro-spraying.-   (5) A lots of spots are formed on the sample chip by repeating the    steps (3) and (4). In this case, the spacer is moved along a    trajectory such that previously deposited spots are not brought into    contact with the spacer.

As illustrated in FIG. 9, spots are successively disposed on the samplechip from its upper left corner to its upper right corner to form afirst row of sample spots. After the spot has been disposed on the upperright corner, the stage is shifted just below the first row, then spotsare disposed in sequence from left to right to form a second row ofsample spots. In this manner, a number of the sample spots can be formedon the sample chip without contacting the spacer 112 with previouslydeposited spots. The trajectory of the movement of the spacer and ashape of the spacer are not limited to the above explained example, widevariety of combination are possible.

INDUSTRIAL APPLICABILITY

The advantages of the device according to present invention aresummarized as follows:

-   (1) The device is applicable to separately prepared DNAs, proteins    and other compounds.-   (2) A great number of the spots can simultaneously be formed in a    short period of time, thereby a large number of sample chips can be    manufactured simultaneously.-   (3) Very minute spots (1–2 μm diameter) can be deposited and sample    chips with a high spot density could be manufacture.-   (4) The spots can be formed using only small amounts of sample    solutions.-   (5) As the result of the above mentioned merits, cost of the chips    (i.e. microarrays) as an end product may be reduced notably lower    than that of conventional chips.

As described above, since the present invention is applicable to varioussubstances such as DNAs and proteins, the present invention is suitablefor various applications as follows:

-   (1) Analysis of genes (gene expression monitoring, gene sequencing,    etc.)-   (2) Analysis of functions of proteins-   (3) Diagnostic products (gene diagnosis, typing of an enzyme,    determination of allergen, identification or typing of infection    fungus, etc.)-   (4) Curing diseases (determination of drugs best fit for respective    conditions of genetic strains or physiological characteristics of    patients, etc.)-   (5) Screenings of drugs and the like (multi-factor high-throughput    screening is possible)-   (6) Analyses (analysis of toxicity of compound, environment,    contamination of microbe of foods, or etc.)

In addition to the above listed applications, it is expected that a muchmore wider variety of applications will be found in the future.

1. A device for manufacturing microarrays comprising: electro-spraying means for electrostatically spraying, in sequence, a plurality of solutions each containing respective one of a plurality of kinds of biologically active samples; supporting means for supporting a plurality of sample chips on which samples contained in the solutions and electrostatically sprayed from said electro-spraying means are deposited; masking means disposed between said electro-spraying means and said supporting means and having holes whose number is equal to the number of the sample chips, a sample being selectively and simultaneously deposited on said sample chips at predetermined corresponding locations, wherein the masking means is formed by sequentially laminating a first insulating layer, an electrically conductive layer, a second insulating layer and a mask layer; and shifting means for shifting said supporting means and said masking means relatively such that the samples are deposited on said plurality of sample chips to manufacture simultaneously a plurality of microarrays.
 2. The device according to claim 1, wherein said electro-spraying means comprises: a single capillary including one or more electrodes; and means for successively feeding to said capillary said plurality of solutions each containing respective one of said plurality of biologically active samples.
 3. The device according to claim 2, wherein the device further comprises means for cleaning said capillary after a sample solution has been electrostatically sprayed and before a next sample solution is electrostatically sprayed.
 4. The device according to claim 2, wherein said electro-spraying means further comprises means for feeding a pressurized air into the capillary to convey the solution to tip of the capillary when the solution is to be electrostatically sprayed.
 5. The device according to claim 2, wherein said electro-spraying means further comprises a guard ring and/or a shield for preventing diffusion of the electrostatically sprayed substances from said capillary.
 6. The device according to claim 2, wherein the device further comprises means for shifting relative position between said capillary and said supporting means and masking means such that the samples are deposited on each of said plurality of sample chips and a plurality of microarrays are manufactured simultaneously.
 7. The device according to claim 1, wherein said electro-spraying means further comprises: means for holding a plurality of multi-capillary cassettes each including a plurality of capillaries each containing respective one of said plurality of sample solutions and having one or more electrodes which are selectively connected with a power source for electro-spraying; and transporting means for successively transporting said plurality of multi-capillary cassettes to an electro-spraying site.
 8. The device according to claim 7, wherein said electro-spraying means further comprises means for applying a pressurized air to all the capillaries in said multi-capillary cassette to convey the sample solutions to tips of capillaries when the solutions are to be electrostatically sprayed.
 9. The device according to claim 7, wherein said holding means further comprises means for controlling a temperature of the plurality of sample solutions contained in said capillaries held in the multi-capillary cassette.
 10. The device according to claim 7, wherein said electro-spraying means further comprises a guard ring and/or a shield for preventing diffusion of the electrostatically prayed substances from said capillaries provided in the multi-capillary cassette.
 11. The device according to claim 7, wherein the device further comprises means for shifting relative position between said multi-capillary cassette and said supporting means and masking means such that the samples are deposited on each of said plurality of sample chips to manufacture a plurality of microarrays simultaneously.
 12. The device according to claim 1, wherein said hole of the masking means is formed such that a size of an opening of the hole facing said electro-spraying means is larger than a size of an opening of the hole facing said holding means.
 13. The device according to claim 1, wherein said masking means comprises a collimating ring for collecting electrostatically sprayed particles toward the hole, said collimating ring being formed integrally with the masking means.
 14. The device according to claim 1, wherein said shifting means for moving said sample chip supporting means and said masking means relatively comprises an X-Y stage or X-Y-Z stage for shifting said supporting means with respect to said masking means.
 15. The device according to claim 1, wherein the device further comprises a plurality of spacers fixed to a surface of said masking means facing to said sample chips at positions near each of the plurality of the holes formed in the masking means.
 16. The device according to claim 1, wherein the device further comprises means for feeding a purified dry air through a casing which surrounds a electro-spraying site.
 17. The device according to claim 1, wherein said sample chips are made of an electrically conductive material or an electrically insulating material coated with an electrically conductive material and are connected to the ground potential. 